Increasing Heart Vascularisation After Myocardial Infarction Using Brain

RESEARCH ARTICLE Increasing heart vascularisation after myocardial infarction using brain natriuretic peptide stimulation of endothelial and WT1+ epicardial cells Na Li, Stephanie Rignault-Clerc, Christelle Bielmann, Anne-Charlotte Bon-Mathier, Tamara De´ glise, Alexia Carboni†, Me´ gane Ducrest, Nathalie Rosenblatt-Velin* Division of Angiology, Heart and Vessel Department, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland Abstract Brain natriuretic peptide (BNP) treatment increases heart function and decreases heart dilation after myocardial infarction (MI). Here, we investigated whether part of the cardioprotective effect of BNP in infarcted hearts related to improved neovascularisation. Infarcted mice were treated with saline or BNP for 10 days. BNP treatment increased vascularisation and the number of endothelial cells in all areas of infarcted hearts. Endothelial cell lineage tracing showed that BNP directly stimulated the proliferation of resident endothelial cells via NPR-A binding and p38 MAP kinase activation. BNP also stimulated the proliferation of WT1+ epicardium-derived cells but only in the hypoxic area of infarcted hearts. Our results demonstrated that these immature cells have a natural capacity to differentiate into endothelial cells in infarcted hearts. BNP treatment increased their proliferation but not their differentiation capacity. We identified new roles for BNP that hold potential for new therapeutic strategies to improve recovery and clinical outcome after MI. *For correspondence: [email protected] Present address: †Ecole Introduction Polytechnique fe´de´ral de Lausanne, Laussane, Switzerland Increased vascularisation supports heart recovery after ischemia. The formation of new vessels in the hypoxic area restores blood flow, provides oxygen and nutriments to the surviving cells, and pro- Competing interests: The motes the migration and engraftment of new cells. While angiogenic inhibition contributes to the authors declare that no development of heart failure in cardiac injury animal models, early heart reperfusion or increased competing interests exist. angiogenesis improves cardiac function and delays the onset of heart failure in patients suffering Funding: See page 24 from cardiac ischemia (Shiojima et al., 2005; Friehs et al., 2006; Tirziu et al., 2007). Received: 15 July 2020 Angiogenesis is the main mechanism of neovascularisation in adult infarcted hearts Accepted: 17 November 2020 (Manavski et al., 2018; Li et al., 2019; Ray et al., 2010). The origin of new endothelial cells (i.e. res- Published: 27 November 2020 ident or infiltrating) as well as the underlying mechanism leading to their proliferation (partial endothelial-to-mesenchymal transition [EndMT] or not) have long been debated. The current consensus is Reviewing editor: Noriaki Emoto, Kobe Pharmaceutical that after myocardial infarction (MI), angiogenesis in the infarct border zone of the heart occurs by University, Japan clonal expansion of pre-existing resident endothelial cells with no EndMT mechanism (Manavski et al., 2018; Li et al., 2019; He et al., 2017). However, the molecular pathway involved Copyright Li et al. This article in myocardial neovascularisation after ischemia remains unknown. Indeed, Payne et al., 2019 is distributed under the terms of recently demonstrated that the developmental VEGFA-MEF2 pathway, which was thought to be the Creative Commons Attribution License, which involved, is in fact impaired in adult ischaemic hearts. permits unrestricted use and Stimulating angiogenesis after MI can improve heart recovery. One complementary therapy could redistribution provided that the be ‘re-activating’ vasculogenesis (i.e. the differentiation of precursor cells into mature endothelial original author and source are cells), a mechanism that occurs in the heart during development but is quiescent in adult hearts. credited. Epicardial cells, and more precisely, cells expressing the Wilms’ tumour 1 transcription factor (WT1) Li et al. eLife 2020;9:e61050. DOI: https://doi.org/10.7554/eLife.61050 1 of 28 Research article Cell Biology Stem Cells and Regenerative Medicine migrate from the epicardium to the myocardium during heart development and then differentiate into coronary endothelial cells, pericytes, smooth muscle cells, and even cardiomyocytes after epi- thelial-to-mesenchymal transition (Zhou et al., 2008; Cano et al., 2016; Smits et al., 2018). Conse- quently, numerous WT1+ cells are found in foetal and neonatal hearts, whereas only a few cells express WT1 in adult hearts (Duim et al., 2016; Duim et al., 2015). In hypoxic adult hearts, numerous proliferating WT1+ cells can be localised in the epicardium near the infarct and border zone, suggesting that hypoxia stimulates either the proliferation of the remaining WT1+ cells or the re-expression of WT1 in cardiac cells (Duim et al., 2015; Balbi et al., 2019; Zhou et al., 2012). WT1+ epicardium-derived cells (EPDCs) remain in a thickened layer on the heart surface without migrating into the myocardium or differentiating into other cell types such as cardiomyocytes or endothelial cells (Zhou et al., 2011). Different treatments (e.g. injections of thy- mosin beta four or human amniotic fluid stem cell secretome) fail to induce WT1+ cell differentiation into endothelial cells in adult hearts after MI (Balbi et al., 2019; Zhou et al., 2012). Despite enhanced WT1+ cell proliferation and higher vessel density in these treated infarcted hearts, no WT1+ cell differentiation into endothelial cells has been detected, implying that WT1+ EPDCs improve neovascularisation in infarcted hearts via paracrine stimulation. It is therefore important to identify soluble factors to increase neovascularisation in the heart after MI. For several years, we have studied the role of brain natriuretic peptide (BNP) in the heart during ageing and after ischaemic damage (Bielmann et al., 2015). BNP is a cardiac hormone belonging to the natriuretic peptide family along with atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP). Although low levels of BNP are co-stored with ANP in atria, high levels of BNP are detected in the ventricle (Potter et al., 2009). BNP is mainly secreted in the ventricles by cardiomyocytes, fibroblasts, and endothelial and precursor cells (Bielmann et al., 2015; Potter et al., 2009; Rosenblatt-Velin et al., 2016). BNP binds to guanylyl cyclase receptors, NPR-A and NPR-B, thus increasing the intracellular cGMP level (Potter et al., 2009). BNP is synthesised in the cell cytoplasm as pre-proBNP precursor, cleaved by furin and corin into proBNP peptide, and then into the biologically active carboxy-terminal BNP peptide (active BNP) and the inactive N-terminal fragment (NT-proBNP) (Yandle and Richards, 2015; Clerico et al., 2012). ProBNP, active BNP, and NT-proBNP peptides are continuously secreted by cardiac cells and ProBNP peptide is the major circulating form of BNP in the plasma of healthy individuals (Shimizu et al., 2002). O-glycosylated proBNP was also detected in the plasma of patients suffering from heart failure (Volpe et al., 2016), and O-glycosylation of proBNP prevents cleavage of this peptide (Volpe et al., 2016). Thus, the balance between proBNP and active BNP is impaired in patients with heart diseases, as they have higher levels of inactive proBNP and reduced levels of active BNP. Patients with heart disease therefore have a deficit in functional active BNP (Chen, 2007). BNP secreted by cardiac cells acts on different organs such as the kidneys (modulating sodium and water excretion), vessels (increasing dilation), fat (increasing lipolysis), and pancreas (modulating insulin secretion) (Rosenblatt-Velin et al., 2016; Volpe et al., 2016). In the heart, a majority of cardiac cells (cardiomyocytes, fibroblasts, endothelial cells) express BNP receptors in physiological and pathological states (Bielmann et al., 2015). BNP treatment after injury protects the heart by reduc- ing fibrosis, cardiomyocyte death, and hypertrophy (D’Souza and Baxter, 2003; Moilanen et al., 2011; Gorbe et al., 2010; Scott et al., 2009; Sun et al., 2010; Wu et al., 2009). In the last few years, we and others reported that BNP supplementation after ischaemic damage promotes the recovery of cardiac function and prevents cardiac remodelling in adult rodent ischaemic hearts (Bielmann et al., 2015; Moilanen et al., 2011). Furthermore, in clinic, a treatment (LCZ696 or Entresto, Novartis) based on inhibition of neprilysin, an enzyme involved in the degrada- tion of the natriuretic peptides, leads to reduced rate of mortality in patients suffering from heart failure with reduced and preserved cardiac contractility or ejection fraction (McMurray et al., 2014). The cellular mechanisms by which BNP exerts its cardioprotective effect are not fully elucidated (Bielmann et al., 2015; Rosenblatt-Velin et al., 2016; Moilanen et al., 2011). Curiously, we observed that BNP stimulates in vitro and in vivo (i.e. in adult infarcted hearts) the proliferation of cardiac non-myocyte cells (NMCs) expressing the stem cell antigen-1 (Sca-1) (Rignault-Clerc et al., 2017). As Sca-1+ cells in adult hearts were reported to be pure endothelial cells (Zhang et al., 2018), we questioned whether BNP modulates endothelial cell fate. Li et al. eLife 2020;9:e61050. DOI: https://doi.org/10.7554/eLife.61050 2 of 28 Research article Cell Biology Stem Cells and Regenerative Medicine Thus, since high BNP levels in plasma are associated with increased

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